Use of a hemoglobin for the preparation of dressings and resulting dressings

ABSTRACT

The invention relates to the use of a haemoglobin for the preparation of dressings and to the resulting dressings.

This application is divisional of U.S. application Ser. No. 12/665,589,filed Jul. 20, 2010, which is a U.S. national phase of InternationalApplication No. PCT/FR2008/00786, filed Jun. 10, 2008, which claimspriority from French Patent application no. 0704312, filed Jun. 18,2007, the disclosure of each of which is hereby incorporated byreference in its entirety.

The present invention relates to the use of a hemoglobin for thepreparation of dressings and to the resulting dressings.

Hemoglobin present in the human or animal bloodstream transports oxygenfrom the lungs to the extremities of the limbs. Oxygen has a very lowsolubility in an aqueous medium, and is therefore distributed in theorganism, very close to the cells, via hemoglobin by virtue of thecapillary network, by diffusion.

When there is an open injury to the skin, the supply of oxygen bydiffusion at the surface of cells is then eliminated.

The skin is a complex tissue which provides elementary protection,sensitivity, thermoregulation and metabolism functions. An interruptionin tissue continuity (wound) can have an effect on each of thesefunctions.

A distinction can be made between two types of wounds, depending on thehealing time: acute wounds and chronic wounds. The healing of the firstoccurs without complication and in less than six weeks. The second aredefined as skin lesions for which the healing time is more than sixweeks.

Chronic wounds group together, inter alia, decubitus ulcers, leg ulcersand “diabetic foot” ulcers.

The decubitus ulcer is a skin, lesion of ischemic origin linked to acompression of the soft tissues between a hard surface and boneprojections.

The leg ulcer is a wound located below the knee, which has notspontaneously healed, and which is in general of venous origin.

The “diabetic foot” is a consequence of vascular and neurologicalcomplications with diabetes, in the feet. These wounds are all directlyor indirectly linked to a problem of oxygenation.

For example, in the case of decubitus ulcers, the compression leads to areduction in blood supply and therefore in O₂ supply (hypoxia) and innutrient supply at the cellular level, which results in ischemia andthen in necrosis. The compression creates a venous obstruction andtherefore a stasis, followed by obstruction of the capillaries and thenof the musculocutaneous arteries.

The ischemia (decrease in blood circulation) results essentially fromthe attack on the vascular system. This results in a reduction in theamounts of O₂ and of nutrients which is responsible for the delay inwound healing. Infection may be superficial, but the risk thereof islinked to deep involvement that may threaten the tissues, sheaths andtendons, and especially the bone structures.

Periodontal diseases are also very common in diabetic patients. Veryoften, the immune system of these patients does not make it possible toeffectively confront a bacterial infection. At the oral level, therefollows an imbalance in the oral flora with a proliferation of anaerobicpathogens.

Numerous external factors are also responsible for this pathologicalcondition, which most of the time is due to a proliferation of anaerobicbacteria.

In order to promote healing, and in particular that of chronic openwounds, several techniques have been developed over the past few years,and in particular the use of hemoglobin alone or in combination with agel.

Thus, patent EP 0 862 440 describes the therapeutic use of hemoglobin ofany type for promoting healing in a patient. The hemoglobin isadministered intravenously and may be natural or chemically modified.However, there are two problems with using natural hemoglobin: firstly,its instability over time and, secondly, the possibility of triggeringan allergic reaction in the patient.

Patent application US 2003/0180365 concerns an externally applicablepreparation containing an oxygen carrier, in which the oxygen carrier isincorporated into, and molecularly dispersed in, a preparation havingthe consistency of a gel, for regeneration of the skin in the case ofoxygen deficiency.

The gel used may be an inorganic gel or an organic gel. The oxygencarrier may be human or animal native hemoglobin or a mixture of saidhemoglobin with a horse, dog or sheep myoglobin.

This gel releases the oxygen-transporting hemoglobin in the skin so asto allow diffusion of the oxygen in the epidermis. This gel does nothave bactericidal properties and cannot therefore be used in the contextof open wounds having too great an exudate, since there is then a needto combat the infection present in the exudate.

Preferably, the hemoglobin is protected from oxidation, i.e. stabilized,in particular with CO. It is specified in this document that hemoglobincan be used without stabilization, but that such a preparation cannot bestored for as long as the stabilized preparation, because of theoxidation of the hemoglobin.

Application US 2004/0022839 describes an externally applicableformulation containing an oxygen carrier, in which the oxygen carrier isincorporated into a lipoid emulsion.

This formulation is intended for skin regeneration in the event ofoxygen deficiency and also cannot be used for the treatment of openwounds.

The oxygen carrier may be hemoglobin and the formulation releases theoxygen-transporting hemoglobin in the skin so as to allow diffusion ofthe oxygen in the epidermis.

The same problems as previously are encountered with this formulation,namely oxidation of the hemoglobin, if the latter is not stabilized withan antioxidant, and the need to use natural cofactors such as2,3-diphosphoglycerate, or non-natural cofactors such as inositolhexaphosphate or mellitic acid, in order to obtain an action onepidermal regeneration.

Application US 2005/0129747 describes the use of an optionally modifiedoxygen carrier for the production of an agent for the external treatmentof open wounds.

The oxygen carrier may be an optionally modified hemoglobin or myoglobinof human or animal origin, and the carrier is used in a solution or byspraying.

The hemoglobin thus deposited in the aqueous barrier of the open woundenables the oxygen to diffuse through this barrier so as to promotehealing.

The limitations of this carrier lie in the oxidation of the nativehemoglobin and, consequently, the need to use antioxidants, cofactors orchemically modified hemoglobin.

Application US 2005/0232953 describes water-in-oil microemulsionscomprising an optionally modified hemoglobin and also antioxidants.

It is specified in this application that the vital cells of theepithelium of the skin are protected from the external environment bythe stratum corneum, which is difficult to penetrate. Consequently, thetwo conditions, according to this application, for an active substanceto bypass the stratum corneum barrier are the following:

-   1) penetration: entry of the substance into the stratum corneum,-   2) permeation: diffusion of the substance from the stratum corneum    to the epidermis.

According to this application, the hemoglobin contained in themicroemulsion penetrates rapidly and deeply into the stratum corneum anddiffuses the oxygen therein.

However, this hemoglobin will rapidly oxidize in the stratum corneum andconsequently requires the presence of an antioxidant, since it is wellknown that, when a hemoglobin is isolated from red blood cells, saidhemoglobin will become oxidized due to the absence of the anti-oxidizingactivity of the enzymes present in the red blood cells (Savitsky J P,Doczi J, Black J & Arnold J D (1978) A clinical safety trial ofstroma-free hemoglobin. Clin Pharmacol Ther 23, 73-80), (Chan W L, TangN L, Yim C C, Lai F M & Tam M S (2000); New features of renal lesioninduced by stroma free hemoglobin, Toxicol Pathol 28, 635-642).

Consequently, one of the objects of the invention is to provide adressing containing hemoglobin which is immobilized and stable in amatrix allowing the treatment of ischemic wounds.

Another object of the invention is to provide a dressing based onhemoglobin which does not require the use of cofactors or ofantioxidants.

Another aspect of the invention is to provide a dressing based onhemoglobin which allows sustained use over time and avoids thetriggering of allergic reactions.

Another object of the invention is to provide cosmetic or pharmaceuticalcompositions comprising hemoglobin in a matrix for the treatment ofischemic wounds and/or of local infections caused by pathogens, inparticular anaerobic pathogens.

Another object of the invention is to provide a method for preparinghemoglobin immobilized in a matrix.

Consequently, the invention relates to the use of a human hemoglobin ora hemoglobin from a vertebrate or invertebrate animal, which isessentially immobilized in a matrix and stable in the matrix, saidmatrix being physiologically acceptable and based on advantageouslypolymerized hydrocolloids, and having a water content of 0 to 98%, as anoxygen carrier in a physiological tissue, in particular a hypoxictissue, requiring an oxygen supply, without the release of thehemoglobin essentially immobilized in the abovementioned matrix beinggreater than 10% by weight.

The expression “hemoglobin essentially immobilized in a matrix” shouldbe understood to mean that the hemoglobin remains in the matrix and isvirtually not released, i.e. freed out of said matrix.

The proportion of hemoglobin released is measured under simulated invivo conditions by calorimetric assay using Drabkin's reagent asexplained in example 5.

The expression “stable in the matrix” means that the hemoglobin is notoxidized in the matrix and that it does not denature.

In particular, the dressing of the invention does not become liquefiedon contact with the wound, thus avoiding release of the hemoglobin.

The matrix represents the medium in which the hemoglobin is immobilizedand is constituted of hydrocolloids, i.e. an attachment agent of plantorigin.

Therefore, the hemoglobin thus trapped in the matrix does not enter intothe skin, whether into the stratum corneum or into the epidermis, whichwill thus allow continual diffusion of the oxygen in the stratumcorneum.

The term “vertebrate animal” denotes a bovine, a mammal such as a pig, asheep, a monkey or a snake, and the term “invertebrate animal” denotesan insect, or an animal belonging to the branch of the annelids.

The term “hypoxic” physiological tissue denotes a tissue that is low inoxygen or has a reduced oxygen level (for example, when the tissuedenotes a wound, the transcutaneous partial pressure of O₂ is less than40 mmHg; Smith B, Devigne L, Slade J, Dooley J & Warren D. Wound RepReg. 4, 224 (1996)).

The hemoglobin immobilized in a matrix constitutes a dressing, thisbeing a term which may subsequently be used to denote said hemoglobinimmobilized in a matrix.

The matrix contains from 0 to 98% of water since it may be in a dryform, i.e. containing from 0 to 5% of water, or in a wet form, i.e.containing from 50% to 98% of water.

In one preferred embodiment, the invention relates to the use of ahemoglobin immobilized in a matrix, as defined above, for thepreparation of a dressing intended for the external treatment of open,deep or chronic wounds, or of periodontal diseases, or for thepreparation of a pharmaceutical composition intended for a gastricdressing or for the preparation of cosmetic compositions.

The open wounds are lesions caused by an external agent and which leadto an opening of the skin.

The deep wounds are wounds in which the muscles and then the bones ororgans may be affected.

The chronic wounds group together in particular decubitus ulcers, legulcers and diabetic foot ulcers (FIG. 1).

The periodontal diseases concern all the support tissues for the teeth:the gum (FIG. 2), the ligament and the alveolar bone.

The term “gastric dressing” should be understood to mean a dressingcapable of forming a film-coating protecting the gastric mucosa andmaking it possible to thus act as a gastric protector for the treatmentin particular of gastritis, of esophageal burns or of meteorism.

The cosmetic compositions are intended for skin regeneration in the caseof oxygen deficiency or for the prevention of this deficiency, and inparticular in the context of degenerative modifications of the skin, orof modifications induced by radiation, by heat or by age, and inparticular wrinkles.

According to another embodiment, the invention relates to the use of ahemoglobin immobilized in a matrix as defined above, in which the matrixis formed from a three-dimensional network defining pores the size ofwhich is from approximately 2 nm to approximately 300 μm, preferentiallyfrom approximately 2 nm to approximately 10 μm, preferentially fromapproximately 2 nm to approximately 1 pin, even more preferentially fromapproximately 5 nm to approximately 200 nm, and preferentially ofapproximately 15 nm.

The size of the pores is advantageously 15 nm so as to be able tocontain the hemoglobin.

The pores are represented in FIG. 4. There are two types of pores in thematrix, large pores measuring approximately 150 μm and much smallerpores, of the order of about ten nanometers, and containing thehemoglobin. These pores are located in the membranes surrounding thelarge pores and contain the hemoglobin.

In one preferred embodiment, in the use of the hemoglobin stabilized inthe matrix, the amount of hemoglobin relative to the total dry weight ofhemoglobin and of matrix is from approximately 0.1% (w/w) toapproximately 60% (w/w), preferentially from approximately 10% (w/w) toapproximately 50% (w/w), preferentially from approximately 15% (w/w) toapproximately 45% (w/w), preferentially from approximately 30% (w/w) toapproximately 40% (w/w), and more preferentially approximately 40%(w/w).

The amount of hemoglobin is herein indicated by dry weight.

If the amount of hemoglobin is less than 0.1%, the oxygen transport willno longer be efficient.

If the amount of hemoglobin is greater than 60%, the dressing thenreleases too much hemoglobin.

An amount of hemoglobin of 40% makes it possible to have the bestcompromise between oxygen transport efficiency and less release.

According to one advantageous embodiment, the invention relates to theuse of a hemoglobin immobilized in a matrix, as defined above, in whichthe percentage water content is from 0% to approximately 98%, andpreferentially approximately 50%.

A percentage water content of 50% represents the best compromise betweenlow release, oxygen transport efficiency and ease of handling of thedressing.

According to one preferred embodiment of the invention, the hemoglobinis chemically modified or crosslinked human or vertebrate-animalhemoglobin.

The vertebrate-animal hemoglobin may be a bovine hemoglobin, or ahemoglobin from a mammal such as a pig, a sheep, a monkey or a serpent.

The hemoglobin may be chemically modified for example with CO so as toobtain a carboxyhemoglobin.

The hemoglobin may be crosslinked by creating a chemical bridge betweentwo of its four polypeptide chains and by linking several hemoglobinmolecules together according to the techniques known in the literature(J. M. Harris (editor): Poly-ethylene glycol chemistry: Biotechnical andBiomedical Application, Plenum, N.Y. et al. 1992).

The crosslinking agents used may be polypropylene glycols orpolyethylene glycols or dialdehydes.

In one preferred embodiment of the invention, the hemoglobin is anextracellular hemoglobin from an invertebrate animal, chosen from thephylum Annelida, and is in particular an extracellular hemoglobinbelonging to marine worms such as Arenicola marina.

In the phylum Annelida, a distinction is made between three classes: thepolychaetes (such as Arenicola marina), the oligochaetes (such as theearthworm Lumbricus terrestris) and the achaetes (such as leeches).

Annelids are segmented protostome animals (having metamers, sometimes avery large number of metamers) in the form of a “worm”. They liveessentially in water (seawater, such as Nereis, or freshwater, such asthe leech), even though some species, such as the earthworms, live inthe soil.

The use of an extracellular hemoglobin which is stable in the matrixmakes it possible to avoid the use of an antioxidant and/or of acofactor in order to function.

The use of an extracellular hemoglobin which is stable in the matrixmakes it possible to involve the intrinsic SOD activity (determined bythe method of Flohé & Ötting; Flohé L, Otting F, Methods Enzymol (1984),105, 93-104) of said hemoglobin, thus providing it with an intrinsicantioxidizing activity, and consequently requiring no antioxidant orcofactor in order to function.

According to one advantageous embodiment of the invention, said matrixis based on chitosan, carrageenans, carboxymethylcellulose or alginates,and in particular sodium alginate or calcium alginate.

The calcium alginate may be obtained by sodium alginate ion exchange, byreaction of sodium alginate with a divalent cation such as calciumchloride, calcium acetate, calcium carbonate or calcium phosphate,preferably calcium chloride.

The chitosan is produced by deacetylation of the chitin present in theexoskeleton of insects and other arthropods (crustaceans, arachnids,etc).

It is an aminopolysaccharide constituted of N-acetyl-D-glucose-2-aminegroups linked to one another by a β-(1,4) linkage.

Carrageenans are linear polysaccharides constituted of more or lesssubstituted galactose molecules. The chain is constituted of subunits,called carrabioses, comprising two galactoses linked via a β-(1,4)linkage. These carrabioses are linked to one another in the chain byα-(1,3) linkages.

Carboxymethylcellulose is a polymer derived from natural cellulose,formed by reaction of cellulose with a base and chloroacetic acid. It isbased on a β-(1,4)-D-glucopyranose structure.

Alginates are polymers of alginic acid (constituted of mannuronic acidand of guluronic acid) obtained from brown algae (Laminariales,Fucales). The sodium alginate is extracted from the algae with sodiumhydroxide and then dried so as to obtain a white powder, having amolecular weight ranging from 32 000 to 200 000 Da.

In one preferred embodiment of the invention, the ratio of said alginateto the total weight of hemoglobin and of matrix is from approximately40% (w/w) to approximately 99% (w/w), preferentially from approximately50% (w/w) to approximately 90% (w/w), preferentially from approximately55% (w/w) to approximately 85% (w/w), preferentially from approximately60% (w/w) to approximately 80% (w/w), and preferentially approximately60% (w/w).

The amount of alginate used herein is indicated as dry weight.

If the ratio is less than 40%, release of the hemoglobin is thenobserved.

If the ratio is greater than 60%, the oxygen transport efficiency isreduced.

According to one advantageous embodiment, the invention relates to theuse of a hemoglobin immobilized in a matrix, as defined above, whichallows the creation of an oxygen gradient through the gel, toward theoxygen-deficient zone.

One of the advantages of the invention lies in the immobilization of thehemoglobin in the matrix with less than 10% release.

This immobilization allows oxygen from the air to be fixed by thehemoglobin through the gel and then the oxygen to be released in thewound, thus resulting in the creation of an oxygen gradient.

By way of example, the affinity of the Arenicola marina hemoglobin inthe dressing is P₅₀=7-8 (the P₅₀ is the PO₂ for which 50% of theArenicola marina hemoglobin is saturated with O₂), i.e. the matrix fixesoxygen from the air if the PO₂ is greater than 8 and releases it if thePO₂ is less than 7 (FIG. 5).

In one preferred embodiment of the invention, the hemoglobin is incombination with one or more elements chosen from cofactors,preservatives such as methyl 4-hydroxybenzoate or propyl4-hydroxybenzoate, antioxidants such as reduced glutathione, ascorbicacid, NADH, human myoglobin or myoglobin of animal origin, or acombination of these elements.

Although the hemoglobin of the invention can function withoutantioxidants or cofactors, it may be advantageous, if necessary, inorder to further increase the lifetime of the dressing, to addantioxidants or preservatives, or cofactors and/or myoglobin, which makeit possible to optimize the functioning of the dressing.

According to one advantageous embodiment, the use of a hemoglobinimmobilized in a matrix, according to the invention, makes it possibleto exert a bactericidal effect on Gram-anaerobic bacteria.

In the context of periodontal diseases (FIG. 2), it is anaerobicbacteria, which are the most pathogenic, that are responsible for thepathological condition (table 1).

These pathogens, which are highly refractory to antibiotic treatments,cause pockets between the gum and the tooth which can lead to bonelesions and loss of the tooth organ.

TABLE 1 List of pathogens responsible for periodontal diseases. Most ofthese bacteria are anaerobic microorganisms. CG PPP LJP GJP RPP AP SAPTAP RP HIV-P NUG Actinobacillus + +++ + ++ + actinomycetemcomitansEikenella corrodens ++ + + + + Capnocytopaga species ++ + + +Porphyromanas gingivalis + ++ +++ + + Prevotella intermedia + + + + ++++ ++ + +++ Bacteroides forsythus + + Prevotella melanogenica + +Fusobacterium nucleatum ++ + + ++ + + Campylobacter rectus + + +++Peptostreptococcus micros + + Treponema species + + ++ + ++ +++Enterobacteria + + Aero-anaerobic Gr+ +++ bacteria CG = chronicgingivitis (or plaque-associated gingivitis), PPP = prepubertalperiodontitis, LJP = localized juvenile periodontitis (or aggressiveperiodontitis), GJP = generalized juvenile periodontitis (or aggressiveperiodontitis), RPP = rapidly progressive periodontitis (or aggressiveperiodontitis), AP = adult periodontitis (or localized/generalizedchronic periodontitis), SAP = active phase of adult periodontitis (oraggressive periodontitis), TAP = tobacco-associated periodontitis (oraggressive periodontitis), RP = refractory periodontitis, HIV-P =HIV-associated periodontitis (necrotizing periodontitis), NUG =necrotizing ulcerative gingivitis (necrotizing periodontitis). Frequencyof isolation of pathogenic bacteria: +, ++, +++.

In fact, when a wound exhibits an exudate, the latter contains bacteriawhich cause an infection and it is then necessary to combat both theexudate and the infection.

The dressing of the invention consequently makes it possible to combatthe infection by draining the exudate; the oxygen then present in thedressing can exert its bactericidal activity, and the oxygen gradientcreated promotes healing.

In another aspect, the invention relates to a dressing comprising ahuman hemoglobin or a hemoglobin from a vertebrate or invertebrateanimal, which is essentially immobilized in a matrix and stable in thematrix, said matrix being physiologically acceptable and based onphysiologically compatible hydrocolloids, having a water content of from0% to 98%, and releasing the hemoglobin contained in the abovementionedmatrix only in a proportion of less than 10%.

The dressing requires the presence of a water content in order tofunction; however, a dressing having a very low water content (less than5% of water) is advantageous since it makes it possible to increase thestorage time.

The dressing defined above may be in the liquid or dry form.

According to one advantageous embodiment, the invention relates to adressing comprising a hemoglobin immobilized in a matrix, as definedabove, in which said matrix is formed from a three-dimensional networkdefining pores, the size of which is from approximately 2 nm toapproximately 300 μm, preferentially from approximately 2 nm toapproximately 10 μm, preferentially from approximately 2 nm toapproximately 1 μm, even more preferentially from approximately 5 nm toapproximately 200 μm, and preferentially approximately 15 nm.

The dressing defined above may be in the liquid or dry form.

In another embodiment, the invention relates to a dressing comprising ahemoglobin immobilized in a matrix, as defined above, in which theamount of hemoglobin relative to the total dry weight of hemoglobin andof matrix is from approximately 0.1% (w/w) to approximately 60% (w/w),preferentially from approximately 10% (w/w) to approximately 50% (w/w),preferentially from approximately 15% (w/w) to approximately 45% (w/w),preferentially from approximately 30% (w/w) to approximately 40% (w/w),and more preferentially approximately 40% (w/w).

The amount of hemoglobin indicated herein corresponds to the dry weightof hemoglobin.

The dressing defined above may be in the liquid or dry form.

According to one advantageous embodiment, the invention relates to adressing comprising a hemoglobin immobilized in a matrix, as definedabove, in which the percentage water content is greater thanapproximately 50% and remaining from approximately 50% to approximately98%, preferentially approximately 95%.

The dressing requires the presence of a water content in order tofunction, otherwise the oxygen gradient cannot be created.

The dressing defined above will be denoted liquid-form dressing.

In another embodiment, the invention relates to a dressing comprising ahemoglobin immobilized in a matrix, as defined above, in which thepercentage water content is less than approximately 5% and remainingfrom approximately 5% to approximately 0%, preferentially approximately2%.

The dressing advantageously contains no water or a very small amount(less than 5% of water), thus allowing prolonged storage thereof andtherefore an extended storage time compared with the wet form. In orderfor the dressing to be functional, it is sufficient to rehydrate it.

The dressing defined above will be denoted dry-form dressing.

According to one advantageous embodiment, the invention relates to adressing comprising a hemoglobin immobilized in a matrix, as definedabove, in which the hemoglobin is chemically modified or crosslinkedhuman or vertebrate-animal hemoglobin.

The dressing defined above may be in the liquid or dry form.

According to another embodiment, the invention relates to a dressingcomprising a hemoglobin immobilized in a matrix, as defined above, inwhich the hemoglobin is an extracellular hemoglobin from an invertebrateanimal, chosen from the phylum Annelida, and in particular anextracellular hemoglobin belonging to marine worms such as Arenicolamarina.

The dressing defined above may be in the liquid or dry form.

In another embodiment, the invention relates to dressing comprising ahemoglobin immobilized in a matrix, as defined above, in which saidmatrix is based on chitosan, carrageenans, carboxymethylcellulose oralginates, and in particular sodium alginate or calcium alginate.

The calcium alginate can be obtained by sodium alginate ion exchange, byreaction of sodium alginate with a divalent cation such as calciumchloride, calcium acetate, calcium carbonate or calcium phosphate,preferably calcium chloride.

The dressing defined above may be in the liquid or dry form.

According to one advantageous embodiment, the invention relates to adressing comprising a hemoglobin immobilized in a matrix, as definedabove, in which said alginate, relative to the total dry weight ofhemoglobin and of matrix, is present in a proportion of fromapproximately 40% (w/w) to approximately 99% (w/w), preferentially fromapproximately 50% (w/w) to approximately 90% (w/w), preferentially fromapproximately 55% (w/w) to approximately 85% (w/w), preferentially fromapproximately 60% (w/w) to approximately 80% (w/w), and preferentiallyapproximately 60% (w/w).

The dressing defined above may be in the liquid or dry form.

According to one advantageous embodiment, the invention relates to adressing comprising a hemoglobin immobilized in a matrix, as definedabove, which allows the creation of an oxygen gradient through the gel,from the oxygenated zone to the hypoxic zone.

The oxygenated zone is the external part of the dressing in contact withthe ambient air, this being the part that will capture the oxygen fromthe air.

The hypoxic zone is the part in contact with the wound and which isdeficient in oxygen.

In another embodiment, the invention relates to a dressing comprising ahemoglobin immobilized in a matrix, as defined above, in which thehemoglobin is in combination with one or more elements chosen fromcofactors, preservatives such as methyl 4-hydroxybenzoate or propyl4-hydroxybenzoate, antioxidants such as reduced glutathione, ascorbicacid, NADH, human myoglobin or myoglobin of animal origin, or acombination of these elements.

The dressing defined above may be in the liquid or dry form.

According to one advantageous embodiment, the invention relates to adressing comprising a hemoglobin immobilized in a matrix, as definedabove, which has a bactericidal effect on anaerobic Gram-bacteria.

The dressing defined above may be in the liquid or dry form.

According to yet another aspect, the invention relates to anintermediate composition in aqueous form, comprising a mixture of ahuman hemoglobin or a hemoglobin from a vertebrate or invertebrateanimal and of a nonpolymerized hydrocolloid, which is physiologicallyacceptable, in solution, in particular in water, or in a physiologicallycompatible buffer.

The intermediate composition defined above will be denoted liquidintermediate composition.

The hydrocolloid is nonpolymerized, i.e. it is an attachment agent ofplant origin, composed of polysaccharides and capable of forming a gel,and chosen from chitosan, carrageenans, carboxymethylcellulose oralginates, such as potassium alginate, lithium alginate, magnesiumalginate or ammonium alginate, and preferably sodium alginate.

According to another aspect, the invention relates to an intermediatecomposition in dried form, having a water content of less than 5%,comprising a mixture of a human hemoglobin or a hemoglobin from avertebrate or invertebrate animal and of a physiologically acceptablehydrocolloid.

The intermediate composition defined above will be denoted dryintermediate composition.

The hydrocolloid is nonpolymerized, as defined above.

According to one advantageous embodiment, the invention relates to anintermediate composition as defined above, in which the amount ofhemoglobin, relative to the total weight of hemoglobin and ofhydrocolloid, is from approximately 0.1% (w/w) to approximately 60%(w/w), preferentially from approximately 10% (w/w) to approximately 50%(w/w), preferentially from approximately 15% (w/w) to approximately 45%(w/w), preferentially from approximately 30% (w/w) to approximately 40%(w/w), and more preferentially approximately 40% (w/w).

The amount of hemoglobin indicated herein is in dry weight.

The intermediate composition defined above may be in the liquid or dryform.

According to one advantageous embodiment, the invention relates to anintermediate composition as defined above, in which the hemoglobin ischemically modified or crosslinked human or vertebrate-animalhemoglobin.

The intermediate composition defined above may be in the liquid or dryform.

In another embodiment, the invention relates to an intermediatecomposition as defined above, in which the hemoglobin is anextracellular hemoglobin from an invertebrate animal, chosen from phylumAnnelida, and in particular an extracellular hemoglobin belonging tomarine worms such as Arenicola marina.

The intermediate composition defined above may be in the liquid or dryform.

According to one advantageous embodiment, the invention relates to anintermediate composition as defined above, in which the hydrocolloid isbased on chitosan, carrageenans, carboxymethylcellulose or alginates,and in particular sodium alginate.

The intermediate composition defined above may be in the liquid or dryform.

In another embodiment, the invention relates to an intermediatecomposition as defined above, in which said alginate, relative to thetotal weight of hemoglobin and of hydrocolloids, is present in aproportion of from approximately 40% (w/w) to approximately 99% (w/w),preferentially from approximately 50% (w/w) to approximately 90% (w/w),preferentially from approximately 55% (w/w) to approximately 85% (w/w),preferentially from approximately 60% (w/w) to approximately 80% (w/w),and preferentially approximately 60% (w/w).

The intermediate composition defined above may be the liquid or dryform.

The amount of alginate indicated herein is in dry weight.

According to one advantageous embodiment, the invention relates to anintermediate composition as defined above, in which the hemoglobin is incombination with one or more elements chosen from cofactors,preservatives such as methyl 4-hydroxybenzoate or propyl4-hydroxybenzoate, antioxidants such as reduced glutathione, ascorbicacid, NADH, human myoglobin or myoglobin of animal origin, or acombination of these elements.

The intermediate composition defined above may be in the liquid or dryform.

According to yet another aspect, the invention relates to apharmaceutical composition comprising, as active substance, a humanhemoglobin or a hemoglobin from a vertebrate or invertebrate animal,which is essentially immobilized in a matrix and stable in the matrix,said matrix being physiologically acceptable, in combination with apharmaceutically acceptable carrier.

According to one advantageous embodiment, the invention relates to apharmaceutical composition as defined above which is in a form that canbe administered topically at a rate of from 0.012 mg/d to 100 mg/d ofactive substance, preferentially from 0.12 mg/d to 100 mg/d, and morepreferentially from 10 mg/d to 20 mg/d, or in a form that can beadministered orally at a rate of from 0.012 mg/kg/d to 100 mg/kg/d ofactive substance, preferentially from 0.12 mg/kg/d to 100 mg/kg/d, andmore preferentially from 10 mg/kg/d to 20 mg/kg/d.

The pharmaceutical compositions that can be administered topically areintended for the treatment of periodontal diseases which concern all thesupport tissues for the teeth: the gum (FIG. 2), the ligament and thealveolar bone, and those that can be administered orally are intendedfor protection of the gastric mucosa, as a gastric protector for thetreatment in particular of gastritis, of esophageal burns or ofmeteorism.

According to yet another aspect, the invention relates to a cosmeticcomposition comprising a human hemoglobin or a hemoglobin from avertebrate or invertebrate animal, which is essentially immobilized in amatrix and stable in the matrix, said matrix being physiologicallyacceptable, in combination with a cosmetically acceptable carrier.

According to one advantageous embodiment, the invention relates to acosmetic composition as defined above, which is in a form that can beadministered topically at a rate of from 0.012 mg/d to 100 mg/d ofactive substance, preferentially from 0.12 mg/d to 100 mg/d, and morepreferentially from 10 mg/d to 20 mg/d.

The cosmetic compositions are intended for regeneration of the skin inthe case of oxygen deficiency or for prevention of this deficiency, andin particular in the context of degenerative modifications of the skin,modifications induced by radiation, by heat or by age, and in particularwrinkles.

According to yet another aspect, the invention relates to a method forpreparing a dressing constituted of a human hemoglobin or a hemoglobinfrom a vertebrate or invertebrate animal, which is essentiallyimmobilized in a matrix based on hydrocolloid and having a water contentof from 0% to 98%, comprising a step of polymerization of saidhydrocolloid contained in a mixture of hydrocolloid and of a hemoglobin,which mixture has a water content of less than 5%, in an aqueoussolution of a divalent or trivalent cation, or a solution containing abridging agent such as dialdehydes.

The compound obtained corresponds to the dressing in wet form containingfrom 50% to 98% of water.

Given the instability of hemoglobin, it was not obvious for thoseskilled in the art that the polymerization of the hydrocolloid can becarried out in the presence of hemoglobin and makes it possible toobtain, as final product, a dressing which conserves all thefunctionalities thereof.

The aqueous solution of divalent or trivalent cation makes it possibleto carry out the polymerization of the hydrocolloid by ion exchange withrespect to the initial hydrocolloid.

The divalent or trivalent cations are chosen from calcium chloride, zincacetate, calcium acetate, calcium carbonate, calcium phosphate, aluminumchloride and glutaraldehyde, preferably calcium chloride, and are usedat a concentration of from 0.1% to 15%, preferentially from 2% to 10%,and more preferentially 10%.

According to one advantageous embodiment, the invention relates to apreparation method, as defined above, in which the mixture of thehydrocolloid and of a hemoglobin having a water content of less than 5%has been prepared by vacuum-drying an aqueous solution of a hydrocolloidand of the hemoglobin.

One of the advantages of the method is that the aqueous solutioncontaining a hydrocolloid and a hemoglobin can be dried beforepolymerization, unlike the techniques normally used, where the dryingoccurs after the polymerization.

The drying before polymerization makes it possible to limit the releaseof the hemoglobin and to obtain a dressing of fine thickness.

Said aqueous solution was prepared by mixing an aqueous solution of ahydrocolloid at a concentration of from 1% to 3% with an aqueoussolution of hemoglobin at a concentration of from 2 to 10 mg/ml.

According to one advantageous embodiment, the invention relates to apreparation method, as defined above, comprising a subsequent step ofdehydration of a dressing having a water content of from approximately50% to 98%, so as to obtain a dressing having a water content of lessthan 5%.

One of the advantages of the method which results in the dressing havinga water content of less than 5% (dry form) lies in the fact that thisdressing can be stored for a longer period of time than the wet formcontaining from 50% to 98% of water (wet form).

According to one advantageous embodiment, the invention relates to apreparation method, as defined above, comprising a subsequent step ofrehydration of the dressing having a water content of less than 5%, soas to obtain a dressing having a water content of from approximately 40%to 70%.

The dressing having a water content of less than 5% (dry form), thestorage product, can advantageously be subsequently rehydrated so as tobe used in the same manner as the wet dressing (wet form) obtaineddirectly.

According to one advantageous embodiment, the invention relates to apreparation method, as defined above, in which the extracellularhemoglobin from an invertebrate animal, chosen from the phylum Annelida,and in particular an extracellular hemoglobin belonging to marine wormssuch as Arenicola marina.

According to another embodiment, the invention relates to a preparationmethod, as defined above, in which the hydrocolloid is based onchitosan, carrageenans, carboxymethylcellulose or alginates, and inparticular sodium alginate or calcium alginate.

According to one advantageous embodiment, the invention relates to apreparation method, as defined above, comprising the following steps:

-   a. mixing a solution containing a hemoglobin and a solution    containing a hydrocolloid, the proportion of hemoglobin, relative to    the total dry weight of hemoglobin and of hydrocolloid, being from    approximately 0.1% (w/w) to approximately 60% (w/w), preferentially    from approximately 10% (w/w) to approximately 50% (w/w),    preferentially from approximately 15% (w/w) to approximately 45%    (w/w), and more preferentially approximately 40% (w/w), so as to    obtain an intermediate composition in aqueous form constituted of a    mixture of hemoglobin and of a hydrocolloid in solution,-   b. vacuum-drying said mixture of hemoglobin and of a hydrocolloid in    solution, so as to obtain a mixture of hemoglobin and of a    hydrocolloid having a water content of less than 5%,-   c. polymerizing said hydrocolloid in said mixture having a water    content of less than 5%, in an aqueous solution of a divalent cation    chosen from calcium chloride, zinc acetate, calcium acetate, calcium    carbonate, calcium phosphate, aluminum chloride or dialdehydes, and    preferentially calcium chloride, at a concentration of from 0.1% to    5%, preferentially from 2% to 10%, and more preferentially 10%, so    as to obtain a dressing having a water content of from 50% to 98%,-   d. optionally, dehydrating by evaporation under vacuum and/or    lyophilizing said dressing having a water content of from 50% to    98%, so as to obtain a storage product having a water content of    less than 5%,-   e. optionally, rehydrating said storage product having a water    content of less than 5%, so as to obtain a dressing having a water    content of from 40% to 70%.

According to another aspect, the invention relates to a dressing havinga water content of from 0% to 98%, as obtained by means of the methoddefined above.

According to another aspect, the invention relates to a combinationproduct containing an intermediate composition in aqueous form asdefined above and an aqueous solution of a divalent or trivalent cation,or an aqueous solution containing a bridging agent such as dialdehydes,for producing a dressing having a water content of from 50% to 98%, byin situ polymerization of said hydrocolloid contained in saidintermediate composition in said aqueous solution.

One of the advantages of the invention is to provide a dressing whichcan be produced in situ (FIG. 6), and which is in particular of use inthe treatment of periodontal diseases, it being possible for thedressing to be applied directly in the buccal cavity, or for thetreatment of deep wounds.

DESCRIPTION OF THE FIGURES

FIG. 1 represents an example of a chronic wound: diabetic patientsuffering from ischemia in the foot.

FIG. 2 represents an attack on the periodontium by anaerobic pathogens,creating pockets between the gum and the tooth organ.

FIG. 3 represents the photograph of the scanning microscopy (JEOL JEM1200=carried out on the external face of the matrix of calcium alginatecontaining Arenicola marina hemoglobin after gold shadowing. Theresolution used here does not make it possible to visualize thehemoglobin.

FIG. 4 represents the photograph of the scanning microscopy carried outon the internal face of the matrix of calcium alginate containingArenicola marina hemoglobin (6 mg/ml) after gold shadowing and showingthe porous appearance of the structure. The resolution used here doesnot make it possible to visualize the hemoglobin.

FIG. 5 shows a Barcroft representation (10 mg/ml Arenicola marinahemoglobin, 15% Ca, 1% sodium alginate, Hepes buffer, pH 7.35) fordetermining the P₅₀.

FIG. 6 represents a photograph of the syringe for producing a dressingin situ. The syringe is constituted of two compartments containing,firstly, the sodium alginate (Satialgine™ US 61) in aqueous solutionwith the hemoglobin and, secondly, an aqueous solution of calciumchloride for the polymerization of the alginate in situ, at the syringeoutlet.

FIG. 7 represents the dialysis polymerization on a “Millipore” Minicellsupport.

FIG. 8 represents the diffusion-chamber polymerization.

FIG. 9 represents the release of Arenicola marina hemoglobin (HbA)contained in a dressing prepared with Satialgine™ US551 alginate(Cargill) at 1% as a function of time.

FIGS. 10, 11 and 12 represent HPLC chromatograms at two wavelengths (280and 414 nm) obtained on samples taken, respectively, at 1 h30, 8 h and24 h, from a solution which is isoionic with respect to human blood (seeexample 5) in which a dressing containing Arenicola marina hemoglobinhas been placed.

FIG. 13 represents the control HPLC chromatogram at the two wavelengths(280 and 414 nm), obtained with a dressing without hemoglobin at 24 h.

EXPERIMENTAL SECTION Example 1 Purification of Hemoglobin from Arenicolamarina

The worms originate from a SeaBait breeding farm. These worms are frozenat −80° C., which causes a hemorrhagic shock and rupture of the wall ofthe worm; hemoglobin extraction is thus facilitated.

The worms, once thawed for 24 h at 4° C. in the presence of theextraction buffer (400 mM NaCl, 2.95 mM KCl, 32 mM MgSO₄, 11 mM CaCl₂,50 mM Hepes, 5 mM ascorbic acid, 10 mM reduced glutathione, pH 7.5,filtered through 0.2 μm) in a proportion of 0.2 ml/g, are centrifuged(4500 g, 4° C., 15 min). The supernatant is recovered and the wormpellet is redispersed in 0.2 ml/g of extraction buffer and centrifugedagain, this being carried out 4 times. The combined supernatants arefiltered under pressure (2 bar) through a 5 μm filter and then a 0.1 μmfilter (Pall filters).

The filtrate can be treated in two ways:

-   -   First method: the filtrate is diafiltered against 5 diavolumes        of storage buffer (90 mM NaCl, 23 mM sodium gluconate, 27 mM        sodium acetate, 5 mM KCl, 1.5 mM MgCl₂, 2.5 mM CaCl₂, pH 7.35,        filtered through 0.2 μm) on a Pellicon XL-1000 kDa        ultrafiltration cassette (Millipore), at 4° C. The retentate is        finally concentrated on this same cassette to ˜100 mg/ml before        sterilizing filtration through a 0.2 μm filter and storage at        −80° C.    -   Second method: the 5 μm filtrate is precipitated at the        isoelectric point of Arenicola marina hemoglobin by adding 50%        by volume of a 0.5N solution of sodium acetate/acetic acid, pH        4.15. After stirring for 30 minutes at 4° C., the solution is        centrifuged (4500 g, 4° C., 15 min). The supernatant is removed        and the pellet (containing the Arenicola marina hemoglobin) is        washed twice against the same volume equivalent of ultrapure        water (4500 g, 4° C., 5 min). The rinsed pellet is redispersed        in the same volume equivalent of the storage buffer with        stirring for 1 h at 4° C. The solution is centrifuged so as to        remove the debris (4500 g, 4° C., 15 min). The supernatant is        filtered under pressure through a 0.1 μm filter (Pall) and then        diafiltered against 2 diavolumes on a Pellicon XL-1000 kDa        cassette to a final concentration of ˜100 mg/ml before        sterilizing filtration through a 0.2 μm filter and storage at        −80° C.

Example 2 Preparation of the Liquid Intermediate Composition

Two types of sodium alginate sold by Cargill: Satialgine™ US 61 andSatialgine™ US 551 EP were used. They conform to European pharmacopeiastandards and are used as an additive, by way of a texturing: thickeningand/or gelling agent, for many therapeutic applications.

They are used at a concentration of from 1 to 3% (w/v) depending on thetype of application and the final texture desired. The sodium alginatepowder is diluted, with magnetic stirring, in MilliQ (MQ) water to thedesired concentration. The higher the alginate concentration, the moredifficult it is to dissolve.

Furthermore, Satialgine™ US 551 EP is more viscous than Satialgine™ US61. It is therefore sometimes necessary to heat (˜50° C.) in order toimprove the dissolution. Once the solution is homogeneous, after a fewhours, it is cooled in ice, before adding the Arenicola marinahemoglobin thereto.

The Arenicola marina hemoglobin is prepared as described previously andstored at −80° C. before use, at a concentration of 100 mg/ml in aphysiological buffer, termed storage buffer, which is calcium-free.

The Arenicola marina hemoglobin is thawed at 4° C. and dissolved in thesodium alginate solution at the concentration of 6 mg/ml. The solutionis then homogenized with magnetic stirring.

Example 3 Preparation of the Dried Intermediate Composition

The solution obtained in example 2 is then dried under an air vacuum andin the presence of silica gel for between 12 and 24 h so as to obtain adried intermediate composition.

Example 4 Polymerization

The dried intermediate composition, which is in the form of a thin film,is immersed in 10 ml of a 1% (w/v) solution of calcium chloride. Thecalcium solution is buffered with 10 mM Hepes (Sigma) at pH 7.0. Thisstep enables the polymerization of the alginate solution and theimmobilization of the Arenicola marina hemoglobin in the alginatematrix.

Several techniques were used to polymerize the solution of sodiumalginate containing hemoglobin. Depending on the method used, it ispossible to obtain various forms of dressing and therefore to envisionthe treatment of various types of wounds or of periodontal infectionslinked to the presence of anaerobic pathogens.

4.1: In Situ Polymerization

Double syringes (Plas-pak) are used for this application. Onecompartment of the syringe (A) is filled with a solution containingsodium alginate (Satialgine™ US 61) at 1% and hemoglobin at 5 mg/ml.

The other compartment (B) is filled with a 1% solution of CaCl₂ in a 10mM Hepes buffer, pH 7.0.

Pressure exerted on the plunger makes it possible to bring the solutionsof the two compartments into contact at the end (C) of the syringe andto polymerize the alginate in solution containing the hemoglobin (D).

4.2: Dialysis Polymerization

The principle of the polymerization is shown in detail in FIG. 7.

The solution of sodium alginate containing the hemoglobin is depositedonto a porous membrane (A) (Minicell, 0.4 μm, Millipore) (HbAm=Arenicolamarina hemoglobin).

The solution can be degassed and dried under vacuum and in the presenceof silica gel (B), before polymerization, for between 12 h and 24 h, orpolymerized as it is.

The polymerization is carried out by immersing the porous membrane (C)in a bath containing a solution containing 1% CaCl₂, 10 mM Hepes, pH7.0, with stirring (D). The calcium diffuses through the membrane duringthe 12 h. The polymerization is carried out at 4° C.

The polymer obtained is then thoroughly rinsed with MilliQ H₂O (E) andthen stored at 4° C. (F). The gel obtained was analyzed by scanningmicroscopy (FIGS. 3 and 4).

It is possible to dry the gel under vacuum by evaporation or bylyophilization, so as to conserve it and to rehydrate it before use (F).

4.3: Diffusion-Chamber Polymerization

The polymerization (FIG. 8) takes place on either side of the aqueoussolution of sodium alginate (1%) and of Arenicola marina hemoglobin (A)which is maintained between two porous membranes (1 μm) in a bathcontaining a solution containing 1% CaCl₂, 10 mM Hepes, pH 7.0, and withstirring (B).

Example 5 Hemoglobin Release

The dressing, prepared according to one of the methods described above,is immersed in 10 ml of a solution that is isoionic with respect tohuman blood (145 mM NaCl, 4 mM KCl, 2 mM MgCl₂, 10 mM Hepes, 2.5 mMCaCl₂, pH 7.35) and the whole is incubated at 34° C. in order tosimulate the physiological medium of the wound.

At regular time periods, an amount of solution (0.5 ml) is removed inorder to assay the released hemoglobin with Drabkin's reagent, readingbeing carried out on a spectrophotometer at 540 nm (colorimetric assay).The concentrations thus obtained in the volume of 0.5 ml are convertedto a total amount of hemoglobin released by the dressing.

FIG. 9 shows the results obtained:

-   -   After 4 h in solution, the dressing has released only 4% of        hemoglobin.    -   After 20 h, the dressing has released only 6% of hemoglobin.    -   After 24 h, only 8% of the hemoglobin contained in the dressing        has been released.

These three measurements confirm that the hemoglobin remains immobilizedin the matrix for more than 24 hours.

An analysis of the structure of the hemoglobin released, by sizeexclusion chromatography (superose 6 column, 0.5 ml/min), was carriedout. Samples were taken at various times (1 h30, 8 h and 24 h) and theoptical density was measured at 280 nm and 414 nm (FIGS. 10, 11, 12).

FIGS. 10, 11 and 12 give the chromatograms obtained and show that thehemoglobin released is a degraded hemoglobin (in dodecamer form, peak at30 minutes), since the absorption peak for native hemoglobin, which isat 21 minutes, is nonexistent.

Consequently, the small percentage of hemoglobin which is released (lessthan 10%) is degraded hemoglobin, thus confirming that the Arenicolamarina hemoglobin remaining in the dressing is stable (otherwise itwould be released).

FIG. 13 gives the chromatogram obtained with a “control” dressingwithout hemoglobin, thus giving the absorption due only to the dressing.

Example 6 Comparison with the Preparation of Application US2003/0180365

The preparation of application US2003/0180365 was prepared as described,by inserting therein 6 mg/ml of Arenicola marina hemoglobin.

The Arenicola marina hemoglobin release tests were carried out asdescribed in example 5.

The formulation thus obtained has the appearance of a very viscous geland, once immersed in the solution which is isoionic with respect tohuman blood, as described in example 5, complete liquefaction of the gelafter 12 h and therefore total release of the Arenicola marinahemoglobin are observed.

The invention claimed is:
 1. A pharmaceutical composition comprising aextracellular Arenicola marina hemoglobin immobilized and stable in aphysiologically acceptable hydrocolloid network, said hydrocolloidnetwork forms a three-dimensional network defining pores, the poresforming a first population of large pores and a second population ofpores wherein the size of the pores in the second population of pores isabout 10 nm, without the release of the hemoglobin immobilized in saidhydrocolloid network being greater than 10%, wherein the Arenicolamarina hemoglobin is not crosslinked, and wherein the hemoglobin iscontained in the second population of pores.
 2. A pharmaceuticalcomposition according to claim 1, which is in a form that can beadministered topically at a rate of from 0.012 mg/d to 100 mg/d ofactive substance or in a form that can be administered orally at a rateof from 0.012 mg/kg/d to 100 mg/kg/d of active substance.
 3. A cosmeticcomposition comprising extracellular Arenicola marina hemoglobinimmobilized and stable in a physiologically acceptable hydrocolloidnetwork, said hydrocolloid network forms a three-dimensional networkdefining pores, the pores forming a first population of large pores anda second population of pores wherein the size of is the pores in thesecond population of pores is about 10 nm, without the release of thehemoglobin immobilized in said hydrocolloid network being greater than10%, wherein the Arenicola marina hemoglobin is not crosslinked, andwherein the hemoglobin is contained in the second population of pores.4. A cosmetic composition according to claim 3, which is in a form thatcan be administered topically at a rate of from 10 mg/d to 20 mg/d.
 5. Acomposition comprising extracellular Arenicola marina hemoglobinimmobilized in a gelled hydrocolloid network, wherein the hydrocolloidnetwork comprises from 0% to 98% by weight of water, and a firstpopulation of large pores and a second population of pores wherein thepores in the second population of pores have a size of about 10 nm; andthe Arenicola marina hemoglobin is contained in the second population ofpores; not crosslinked; and wherein the hemoglobin is present in anamount of about 0.1% w/w to about 60% w/w relative to the total dryweight of the hemoglobin and the hydrocolloid network.
 6. Thecomposition according to claim 5, wherein the composition is in the formof a dressing capable of covering a wound on skin.
 7. A method for theexternal treatment of open, deep or chronic wounds, or of periodontaldiseases, or of a gastric wounds or of tissues, the method comprisingadministering a pharmaceutical composition according to claim 1 to awound or diseased tissue.
 8. The method of claim 7, wherein the amountof hemoglobin, relative to the total dry weight of hemoglobin and ofhydrocolloid network, is from 0.1% (w/w) to 60% (w/w).
 9. The method ofclaim 7, wherein the percentage water content is from greater than 50%.10. The method of claim 7, wherein said hydrocolloid network is based onchitosan, carrageenans, carboxymethylcellulose or alginates.
 11. Themethod of claim 10, wherein said hydrocolloid network is based oncalcium alginate, and wherein the amount of hemoglobin present in thehydrocolloid network, relative to the total weight of hemoglobin and ofthe hydrocolloid network is 55% (w/w) to 85%.
 12. The compositionaccording to claim 5, wherein the amount of Arenicola marina hemoglobin,relative to the total dry weight of Arenicola marina hemoglobin and ofthe hydrocolloid network, is from 15% w/w to 45% w/w.
 13. Thecomposition according to claim 5, wherein the water content is from 95%w/w to 98% w/w.
 14. The composition according to claim 5, wherein thewater content is less than 5% w/w.
 15. The composition according toclaim 5, wherein said hydrocolloid network comprises a hydrocolloidselected from the group consisting of chitosan, carrageenans,carboxymethylcellulose and alginates.
 16. The composition according toclaim 5, wherein the hydrocolloid network is based on an alginate andsaid alginate is calcium alginate, and wherein the amount of Arenicolamarina hemoglobin relative to the total dry weight of hemoglobin and ofthe hydrocolloid network, is 40% w/w to 60% w/w.
 17. The compositionaccording to claim 5, wherein the hydrocolloid network comprises agel-forming polysaccharide.